ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking

The effect of angiotensin II (ANG II) or arginine vasopressin (AVP) alone or plus atrial natriuretic peptide (ANP) on H+-ATPase subcellular vesicle trafficking was investigated in MDCK cells following intracellular pH (pHi) acidification by exposure to20 mMNH4Cl for 2 min in a Na+-free solution containing Schering 28080, conditions under which H+-AT-Pase is the only cell mechanism for pHi recovery. Using the acridine orange fluorescent probe (5mM) and confocal microscopy, the vesicle movement was quantified by determining, for each experimental group, the mean slope of the line indicating the changes in apical/basolateral fluorescence density ratio over time during the first 5.30 min of the pHi recovery period. Under the control conditions, the mean slope was 0.079 ± 0.0033 min-1 (14) and it increased significantly with ANG II [10-12 and 10-7 M, respectively to 0.322 ± 0.038 min-1 (13) and 0.578 ± 0.061 min-1 (12)] or AVP [10-12 and 10-6 M, respectively to 0.301 ± 0.018 min-1 (12) and 0.687 ± 0.049 min-1 (11)]. However, in presence of ANP (10-6 M, decreases cytosolic free calcium), dimethyl-BAPTA/AM (5 × 10-5 M, chelates intracellular calcium) or colchicine (10-5 M, 2-h preincubation; inhibits microtubule-dependent vesicular trafficking) alone or plus ANG II or AVP the mean slopes were similar to the control values, indicating that such agents blocked the stimulatory effect of ANG II or AVP on vesicle trafficking. The results suggest that the pathway responsible for the increase in cytosolic free calcium and the microtu-bule-dependent vesicular trafficking are involved in this hormonal stimulating effect. Whether cytosolic free calcium reduction represents an important direct mechanism for ANP impairs the dose-dependent stimulatory effect of ANG II or AVP on H+-ATPase subcellular vesicle trafficking, or is a side effect of other signaling pathways which will require additional studies.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Pesquisas (CNPq

Incremento na atividade da H+ -ATPase do tipo vacuolar induzido pelo estímulo do receptor extracelular sensível ao cálcio (CaSR) em um modelo de célula intercalar renal (MDCK C11)

Orientador : Prof. Dr. Ricardo Fernandez PerezTese (doutorado) - Universidade Federal do Paraná, Setor de Ciências Biológicas, Programa de Pós-Graduação em Fisiologia. Defesa: Curitiba, 27/10/2017Inclui referências : f. 52-58Resumo: Alguns estudos já demonstraram que o estímulo do Receptor Sensível ao Cálcio Extracelular, o CaSR, é capaz de a secreção de prótons no néfron proximal e distal. Tendo em vista que a acidificação do fluido tubular no ducto coletor é fundamental para evitar a formação de cálculos, o objetivo deste trabalho foi investigar a interação do CaSR com o transporte de prótons num modelo de células intercalares do ducto coletor de mamíferos, as células MDCK C11 (Madin-Darby canine kidney cell). As células MDCK C11 foram cultivadas em meio MEM (Minimum Essential Medium) e suplementadas com 10% de SBF (Soro Bovino Fetal). A atividade bioquímica da H+-ATPasev, sensível a concanamicina 10-8 M, foi determinada por um método colorimétrico modificado do descrito por Fisk-Subarow. Alterações do cálcio intracelular foram determinadas por microscopia de fluorescência, utilizando-se o marcador FLUO-4 10 ?M. O agonista não específico de CaSR, gadolíneo 300 ?M, e o agonista específico de CaSR, R-568 0,5 e 1 ?M, incrementaram de forma significativa a atividade da H+-ATPasev em relação à atividade basal da bomba. O agonista não específico de CaSR, neomicina 200?M, incrementou a atividade da H+-ATPasev, de forma não significativa. Foi observado que o incremento do íon cálcio no meio extracelular estimulou a atividade da H+ - ATPasev de forma dose-dependente. O antagonista seletivo de CaSR, NPS 2143 150 nM, impediu o aumento da atividade da bomba promovido pelo Ca2+o 1 mM. O inibidor das fosfolipases A e C, U-73122 5x10-8 M, reverteu parcialmente o incremento na atividade da bomba observado na presença de gadolíneo 300 ?M. O ativador de proteína quinase C aumentou a atividade da H+-ATPasev em relação à basal, mas não de forma significativa. O agonista específico de CaSR, R-568 1 ?M, promoveu um aumento significativo do cálcio intracelular nas células MDCK C11, o qual persistiu mesmo com a depleção dos estoques intracelulares de Ca2+ com tapsigargina 0,1 ?M. Entretanto, na ausência de cálcio extracelular este aumento não foi observado, o que indica que o aumento do Ca2+ intracelular induzido pela ativação do CaSR é dependente da entrada de Ca2+ do meio extracelular. Destes resultados é possível concluir que o estímulo do CaSR num modelo de célula intercalar renal provoca um incremento na concentração intracelular de cálcio e na atividade bioquímica da H+-ATPasev, sugerindo uma possível interação entre o metabolismo de cálcio e os mecanismos de secreção de prótons nos túbulos renais. Palavras-chave: H+ -ATPase vacuolar; CaSR; células MDCK C11.Abstract: Some studies have already shown that the stimulus of a Calcium-Sensing Receptor, CaSR, is able to increase protons secretions in proximal and distal nephron. Considering that fluid acidification in collecting duct is fundamental to avoid stone formation, the aim of this study was to investigate the interaction of CaSR with proton transport in a mammalian intercalated cell model, C11-MDCK (Madin-Darby canine kidney cell) cells. C11-MDCK cells were cultured in MEM (Minimum Essential Medium) and supplemented with 10% FBS (Fetal Bovine Serum).The concanamycin-sensitive activity of the H+ -ATPasev was determined by a colorimetric method modified of that described by Fisk-Subarow. Changes in intracellular calcium levels were determined by fluorescence microscopy using 10 ?M FLUO-4 dye. The non-specific CaSR agonist, gadolinium 300?M, and the specific CaSR agonist, 0,5 and 1 ?M R-568, increased significantly the activity of H+-ATPasev in relation to the basal activity of the pump. The non-specific CaSR agonist, 200 ?M neomycin, increased the activity of H+-ATPasev but not significantly. It was observed that the increase of extracellular calcium stimulated the activity of H+ -ATPasev in a dose-dependent manner. The selective CaSR antagonist, 150 nM NPS 2143, prevented the increase of pump activity promoted by 1 mM Ca2+. The phospholipase A and C inhibitor, 5x10-8 M U-73122, partially reversed the increase in pump activity observed in the presence of 300?M gadolinium. Protein kinase C activator increased the activity of H+-ATPasev relative to baseline, but not significantly. The specific CaSR agonist, R-568 1 ?M, promoted a significant increase of intracellular calcium in C11-MDCK cells, which persisted even with the depletion of intracellular Ca2+ stores with 0,1 ?M thapsigargin. However, in the absence of extracellular calcium this increase was not observed, which indicates that the increase of intracellular Ca2 + induced by CaSR activation is dependent on the input of Ca2+ from the extracellular medium Ca2+. From these results it can be concluded that the CaSR stimulus on a model of intercalated cells increase intracellular calcium concentration and biochemical activity of H+ -ATPasev, suggesting a possible interaction between calcium metabolism and the mechanisms of proton secretion in renal tubules. Keywords: vacuolar H+ -ATPase; CaSR; C11-MDCK cells

Influência do receptor sensível a cálcio extracelular (CaSR) sobre a atividade da H+-ATPase Vacuolar em células do túbulo proximal

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